Shielding the front-strand β3 of the von Willebrand factor A1 domain inhibits its binding to platelet glycoprotein Ibα

Abstract

Platelet adhesion to damaged vessel wall and shear-induced platelet aggregation necessitate binding of the von Willebrand factor (VWF) A1 domain to platelet GPIb␣. Blocking this interaction represents a promising approach to the treatment of arterial thrombosis. Comparison of amino acid sequences of the VWF A1 domain in several species, expressing VWF recognized by the blocking monoclonal antibody AJvW-2, suggested 9 residues (His563, Ile566, Asp570, Ala581, Val584, Ala587, Arg616, Ala618, and Met622) to contribute to the epitope for AJvW-2 or to be part of the GPIb␣-binding site. Glutathione-S-transferase (GST)-human VWF A1 fusion proteins, in which these amino acids were mutated to their murine counterparts, were tested for their capacity to bind AJvW-2 or heparin, to interfere with botrocetin-or ristocetin-mediated VWF binding to GPIb, or to induce flow-dependent platelet tethering in a perfusion chamber. Thus, mutations His563Arg, Ile566Leu, Asp570Ala, and Ala587Thr, clustered on the outer surface of the A1 domain, dramatically impaired binding of AJvW-2 to A1. The His563Arg, Ile566Leu, and Asp570Ala mutations also impaired the binding of heparin, which competes with AJvW-2 for binding to A1. Perfusion studies revealed that His563, Ile566, Asp570, Arg616, and Ala618 take part in GPIb␣ binding, their mutation-impairing platelet recruitment. In agreement with the surface distribution of VWF type 2M mutations, this study demonstrates overlapping of the epitope for AJvW-2 and the GPIb␣-binding site, located around the front pocket of the A1 domain and defined by strands ␤3, ␤4, and helix ␣3, and it provides a mechanistic basis for VWF neutralization by this antibody. (Blood.

Clumping factor A, von Willebrand factor‐binding protein and von Willebrand factor anchor Staphylococcus aureus to the vessel wall

“…Recombinant His 6 ‐ClfA 1 ‐520 was (ClfA, Clumping factor A) cloned with plasmid pET15 and purified from E. coli BL21 (DE3) using Ni‐NTA chromatography as previously described . Glutathione‐S‐transferase (GST)–VWF A1‐domain proteins were produced as described before .…”

A novel mechanism of sustained platelet αIIbβ3 activation via PEAR1

“…Recombinant fusion proteins were purified using glutathione sepharose beads as previously described. 9 Polyclonal anti-PEAR1 EMI domain antibodies were raised in our laboratory in rabbits, immunized with GST-EMI, referred to as EMI Ab. Treatment of these antibodies with papain yielded Fab fragments, isolated via protein A chromatography and referred to as EMI F(ab).…”

Inhibition of Platelet Glycoprotein Ib and Its Antithrombotic Potential

“…Binding of the A1-domain to GPIb is not dependent on the presence of a divalent cation. The GPIb binding site is located around the front pocket of the A1-domain and consists of the 3 and 4 strand, together with the helix 3 [51]. The A1-domain contains furthermore regulatory N-and C-terminal arms, wrapping across the lower surface, which inhibit the GPIb binding [52].…”